Native Neuropeptides Research Articles

Conformational and functional changes of the native neuropeptide somatostatin occur in the presence of copper and amyloid-β.

The progression of neurodegenerative disorders can lead to impaired neurotransmission; however, the role of pathogenic factors associated with these diseases and their impact on the structures and functions of neurotransmitters have not been clearly established. Here we report the discovery that conformational and functional changes of a native neuropeptide, somatostatin (SST), occur in the presence of copper ions, metal-free amyloid-β (Aβ) and metal-bound Aβ (metal-Aβ) found as pathological factors in the brains of patients with Alzheimer's disease. These pathological elements induce the self-assembly of SST and, consequently, prevent it from binding to the receptor. In the reverse direction, SST notably modifies the aggregation profiles of Aβ species in the presence of metal ions, attenuating their cytotoxicity and interactions with cell membranes. Our work demonstrates a loss of normal function of SST as a neurotransmitter and a gain of its modulative function against metal-Aβ under pathological conditions.

Identification and characterization of expression profiles of neuropeptides and their GPCRs in the swimming crab, Portunus trituberculatus.

Neuropeptides and their G protein-coupled receptors (GPCRs) regulate multiple physiological processes. Currently, little is known about the identity of native neuropeptides and their receptors in Portunus trituberculatus. This study employed RNA-sequencing and reverse transcription-polymerase chain reaction (RT-PCR) techniques to identify neuropeptides and their receptors that might be involved in regulation of reproductive processes of P. trituberculatus. In the central nervous system transcriptome data, 47 neuropeptide transcripts were identified. In further analyses, the tissue expression profile of 32 putative neuropeptide-encoding transcripts was estimated. Results showed that the 32 transcripts were expressed in the central nervous system and 23 of them were expressed in the ovary. A total of 47 GPCR-encoding transcripts belonging to two classes were identified, including 39 encoding GPCR-A family and eight encoding GPCR-B family. In addition, we assessed the tissue expression profile of 33 GPCRs (27 GPCR-As and six GPCR-Bs) transcripts. These GPCRs were found to be widely expressed in different tissues. Similar to the expression profiles of neuropeptides, 20 of these putative GPCR-encoding transcripts were also detected in the ovary. This is the first study to establish the identify of neuropeptides and their GPCRs in P. trituberculatus, and provide information for further investigations into the effect of neuropeptides on the physiology and behavior of decapod crustaceans.

Identification of putative neuropeptidergic signaling systems in the spiny lobster, Panulirus argus.

Members of the decapod infraorder Achelata, specifically species from the genus Panulirus, have storied histories as models for investigating the basic principles governing the generation, maintenance, and modulation of rhythmic motor behavior, including modulation by locally released and circulating peptides. Despite their contributions to our understanding of peptidergic neuromodulation, little is known about the identity of the native neuropeptides and neuronal peptide receptors present in these crustaceans. Here, a Panulirus argus nervous system-specific transcriptome was used to help fill this void, providing insight into the neuropeptidome and neuronal peptide receptome of this species. A neuropeptidome consisting of 266 distinct peptides was predicted using the P. argus assembly, 128 having structures placing them into a generally recognized arthropod peptide family: agatoxin-like peptide, allatostatin A (AST-A), allatostatin B, allatostatin C, bursicon, CCHamide, crustacean cardioactive peptide, crustacean hyperglycemic hormone/molt-inhibiting hormone, diuretic hormone 31 (DH31), ecdysis-triggering hormone (ETH), FMRFamide-like peptide (FLP), glycoprotein hormone (GPH), GSEFLamide, inotocin, leucokinin, myosuppressin, natalisin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, periviscerokinin, pigment-dispersing hormone, pyrokinin, red pigment-concentrating hormone, RYamide, short neuropeptide F (sNPF), SIFamide, sulfakinin, tachykinin-related peptide (TRP), and trissin. Twenty-five putative neuronal receptors, encompassing 15 peptide groups, were also identified from the P. argus transcriptome: AST-A, bursicon, CCHamide, DH31, diuretic hormone 44, ETH, FLP, GPH, inotocin, insulin-like peptide, myosuppressin, natalisin, periviscerokinin, sNPF, and TRP. Collectively, the reported data provide a powerful resource for expanding studies of neuropeptidergic control of physiology and behavior in members of the genus Panulirus specifically, and decapods generally.

Activity-evoked and spontaneous opening of synaptic fusion pores

Synaptic release of neuropeptides packaged in dense-core vesicles (DCVs) regulates synapses, circuits, and behaviors including feeding, sleeping, and pain perception. Here, synaptic DCV fusion pore openings are imaged without interference from cotransmitting small synaptic vesicles (SSVs) with the use of a fluorogen-activating protein (FAP). Activity-evoked kiss and run exocytosis opens synaptic DCV fusion pores away from active zones that readily conduct molecules larger than most native neuropeptides (i.e., molecular weight [MW] up to, at least, 4.5 kDa). Remarkably, these synaptic fusion pores also open spontaneously in the absence of stimulation and extracellular Ca2+ SNARE perturbations demonstrate different mechanisms for activity-evoked and spontaneous fusion pore openings with the latter sharing features of spontaneous small molecule transmitter release by active zone-associated SSVs. Fusion pore opening at resting synapses provides a mechanism for activity-independent peptidergic transmission.

Imaging neuropeptide release at synapses with a genetically engineered reporter.

Research on neuropeptide function has advanced rapidly, yet there is still no spatio-temporally resolved method to measure the release of neuropeptides in vivo. Here we introduce Neuropeptide Release Reporters (NPRRs): novel genetically-encoded sensors with high temporal resolution and genetic specificity. Using the Drosophila larval neuromuscular junction (NMJ) as a model, we provide evidence that NPRRs recapitulate the trafficking and packaging of native neuropeptides, and report stimulation-evoked neuropeptide release events as real-time changes in fluorescence intensity, with sub-second temporal resolution.

Aplysia allatotropin-related peptide and its newly identified d-amino acid–containing epimer both activate a receptor and a neuronal target

l- to d-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the d-residue is critical for the biological function of the resulting d-amino acid-containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-l-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a d-amino acid-containing peptide (d2-ATRP) and identified a specific G protein-coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-l-residue analogs, both l-ATRP and d2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d2-ATRP was much more stable than its all-l-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the l- to d-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that l- to d-residue isomerization can occur even in an all-l-residue peptide with a known biological activity and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.

Release kinetics of somatostatin from self-assembled nanostructured hydrogels.

Somatostatin-14 is a native neuropeptide with widespread functions in the body. Self-assembly of somatostatin-14 into amyloid-like nanofibrils has been previously demonstrated in aqueous media. We here hypothesize that the somatostatin nanofibrils can form a stable depot that release monomers in a controlled manner. This study aims to investigate if somatostatin monomers are released from physical hydrogels formed in water and in the presence of electrolytes. The release kinetics of the somatostatin monomers is investigated for the first time. This is correlated with the rheological properties of the hydrogels formed. We demonstrate that at the concentrations tested, there is release of somatostatin monomers from the hydrogels following a novel hybrid model of zero-order and first-order release. In the presence of electrolytes, somatostatin hydrogels demonstrated higher elastic moduli (G') which correlates to the narrower and higher density of nanofibrils observed with TEM. The presence of electrolytes resulted in a slower release of the somatostatin monomers and in a lower cumulative percentage released over 48 hrs. It is questionable that the concentrations released will be therapeutically effective. However, self-assembled somatostatin hydrogels have the potential to act as a depot for ocular drug delivery.

Positron Emission Tomography Assessment of the Intranasal Delivery Route for Orexin A.

Intranasal drug delivery is a noninvasive drug delivery route that can enhance systemic delivery of therapeutics with poor oral bioavailability by exploiting the rich microvasculature within the nasal cavity. The intranasal delivery route has also been targeted as a method for improved brain uptake of neurotherapeutics, with a goal of harnessing putative, direct nose-to-brain pathways. Studies in rodents, nonhuman primates, and humans have pointed to the efficacy of intranasally delivered neurotherapeutics, while radiolabeling studies have analyzed brain uptake following intranasal administration. In the present study, we employed carbon-11 radioactive methylation to assess the pharmacokinetic mechanism of intranasal delivery of Orexin A, a native neuropeptide and prospective antinarcoleptic drug that binds the orexin receptor 1. Using physicochemical and pharmacological analysis, we identified the methylation sites and confirmed the structure and function of methylated Orexin A (CH3-Orexin A) prior to monitoring its brain uptake following intranasal administration in rodent and nonhuman primate. Through positron emission tomography (PET) imaging of [11C]CH3-Orexin A, we determined that the brain exposure to Orexin A is poor after intranasal administration. Additional ex vivo analysis of brain uptake using [125I]Orexin A indicated intranasal administration of Orexin A affords similar brain uptake when compared to intravenous administration across most brain regions, with possible increased brain uptake localized to the olfactory bulbs.

Anxiogenic drug administration and elevated plus-maze exposure in rats activate populations of relaxin-3 neurons in the nucleus incertus and serotonergic neurons in the dorsal raphe nucleus

Anxiety is a complex and adaptive emotional state controlled by a distributed and interconnected network of brain regions, and disruption of these networks is thought to give rise to the behavioral symptoms associated with anxiety disorders in humans. The dorsal raphe nucleus (DR), which contains the majority of forebrain-projecting serotonergic neurons, is implicated in the control of anxiety states and anxiety-related behavior via neuromodulatory effects on these networks. Relaxin-3 is the native neuropeptide ligand for the Gi/o-protein-coupled receptor, RXFP3, and is primarily expressed in the nucleus incertus (NI), a tegmental region immediately caudal to the DR. RXFP3 activation has been shown to modulate anxiety-related behavior in rodents, and RXFP3 mRNA is expressed in the DR. In this study, we examined the response of relaxin-3-containing neurons in the NI and serotonergic neurons in the DR following pharmacologically induced anxiety and exposure to an aversive environment. We administered the anxiogenic drug FG-7142 or vehicle to adult male Wistar rats and, 30min later, exposed them to either the elevated plus-maze or home cage control conditions. Immunohistochemical detection of c-Fos was used to determine activation of serotonergic neurons in the DR and relaxin-3 neurons in the NI, measured 2h following drug injection. Analysis revealed that FG-7142 administration and exposure to the elevated plus-maze are both associated with an increase in c-Fos expression in relaxin-3-containing neurons in the NI and in serotonergic neurons in dorsal and ventrolateral regions of the DR. These data are consistent with the hypothesis that relaxin-3 systems in the NI and serotonin systems in the DR interact to form part of a network involved in the control of anxiety-related behavior.

Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor

Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent since mutant Y1 receptors unable to recruit β-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gβ/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.

Neuropeptide discovery in the Araneae (Arthropoda, Chelicerata, Arachnida): Elucidation of true spider peptidomes using that of the Western black widow as a reference

The public deposition of large transcriptome shotgun assembly (TSA) datasets for the Araneae (true spiders) provides a resource for determining the structures of the native neuropeptides present in members of this chelicerate order. Here, the Araneae TSA data were mined for putative peptide-encoding transcripts using the recently deduced neuropeptide precursors from the Western black widow Latrodectus hesperus as query templates. Neuropeptide-encoding transcripts from five spiders, Latrodectus tredecimguttatus, Stegodyphus mimosarum, Stegodyphus lineatus, Stegodyphus tentoriicola and Acanthoscurria geniculata, were identified, including ones encoding members of the allatostatin A, allatostatin B, allatostatin C, allatotropin, CAPA/periviscerokinin/pyrokinin, crustacean cardioactive peptide, crustacean hyperglycemic hormone/ion transport peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FMRFamide-like peptide (FLP), GSEFLamide, insulin-like peptide, orcokinin, proctolin, short neuropeptide F, SIFamide, sulfakinin and tachykinin-related peptide (TRP) families. A total of 156 distinct peptides were predicted from the precursor proteins deduced from the S. mimosarum transcripts, with 65, 26, 21 and 12 peptides predicted from those deduced from the A. geniculata, L. tredecimguttatus, S. lineatus and S. tentoriicola sequences, respectively. Among the peptides identified were variant isoforms of FLP, orcokinin and TRP, peptides whose structures are similar to ones previously identified from L. hesperus. The prediction of these atypical peptides from multiple spiders suggests that they may be broadly conserved within the Araneae rather than being species-specific variants. Taken collectively, the data described here greatly expand the number of known Araneae neuropeptides, providing a foundation for future functional studies of peptidergic signaling in this important Chelicerate order.

Brain neuropeptides in central ventilatory and cardiovascular regulation in trout

Many neuropeptides and their G-protein coupled receptors (GPCRs) are present within the brain area involved in ventilatory and cardiovascular regulation but only a few mammalian studies have focused on the integrative physiological actions of neuropeptides on these vital cardio-respiratory regulations. Because both the central neuroanatomical substrates that govern motor ventilatory and cardiovascular output and the primary sequence of regulatory peptides and their receptors have been mostly conserved through evolution, we have developed a trout model to study the central action of native neuropeptides on cardio-ventilatory regulation. In the present review, we summarize the most recent results obtained using this non-mammalian model with a focus on PACAP, VIP, tachykinins, CRF, urotensin-1, CGRP, angiotensin-related peptides, urotensin-II, NPY, and PYY. We propose hypotheses regarding the physiological relevance of the results obtained.

Anticancer activity of a peptide combination in gastrointestinal cancers targeting multiple neuropeptide receptors

A novel peptide combination consisting of four synthetic neuropeptide analogs of Vasoactive Intestinal Peptide (VIP), Bombesin, Substance P and Somatostatin has been found to have potent anticancer activity in vitro and in vivo. The receptors of these four neuropeptides are known to be over expressed in various cancers. We have found the presence of native neuropeptides in the culture supernatant of the primary tumor cells of human colon adenocarcinomas. It was further demonstrated by receptor-ligand assays that not only do these tumor cells synthesize and secrete four peptide hormones but also possess specific high affinity receptors on their surface. Screening a large panel of analogs to the four peptide hormones on tumor cell proliferation led to the identification of four cytotoxic analogs, the combination of which was code-named DRF7295. The design and synthesis of the peptide analogs have been described in this paper. In vitro anticancer activity of DRF7295 was studied in a large panel of human tumor cells. Gastrointestinal tumor cells of the colon, pancreas and duodenum were found to be most sensitive to DRF7295 with moderate activity seen in glioblastoma, prostate, leukemia and those of oral cancer cells. Efficacy studies in xenograft models of colon and duodenum resulted in T/C% of less than 40%, which is indicative of strong tumor regressing potential of DRF7295 in gastrointestinal cancers. Acute and long-term toxicity studies as well as safety pharmacology studies conducted indicate the safety of the drug upon systemic administration with no significant adverse pharmacological effects.

Spontaneous fibrillation of the native neuropeptide hormone Somatostatin-14

Natural Somatostatin-14 is a small cyclic neuropeptide hormone with broad inhibitory effects on endocrine secretions. Here we show that natural Somatostatin-14 spontaneously self-assembles in water and in 150mM NaCl into liquid crystalline nanofibrils, which follow characteristic structural features of amyloid fibrils. These non-covalent highly stable structures are based on the Somatostatin native backbone conformation and are formed under non-denaturing conditions. Our results support the hypothesis that self-assembly into amyloid fibrils is a generic property of the polypeptide chain under appropriate conditions. Given recent advances on the mechanisms of biological storage and sorting modes of peptide/protein hormones into secretory granules, we propose that Somatostatin-14 fibrillation could be relevant to the regulated secretion pathway of this neuropeptide hormone. Such a hypothesis is consistent with the emerging concept of the existence of non-disease related but functional amyloids.

Neuropeptide Y as a partial agonist of the Y1 receptor

In absence of receptor cycling, human/rat neuropeptide Y was found to persistently occupy the guinea pig neuropeptide Y Y1 receptors expressed on the surface of Chinese hamster ovary (CHO) cells (IC 50 ∼ 8 nM); a lasting occupancy was also evident with active receptor cycling. A similar blockade was obtained with the human neuropeptide Y Y1 receptor (in CHO or SK-N-MC cells). Peptidic antagonists GR238118 (1229U91) and VD-11 blocked the Y1 receptor in the same molarity range. A neuropeptide Y-related Y1 agonist, (Leu 31Pro 34) human neuropeptide Y, also strongly adhered to the Y1 site. Similar blockade-like occupancy by neuropeptide Y was found with particulates from Y1-expressing CHO cells, and with native neuropeptide Y Y1 receptors of rat synaptosomes. Peptide YY and a related Y1-selective agonist, (Leu 31Pro 34) human peptide YY, showed a much less stable binding to the neuropeptide Y Y1 receptor with either the intact cells or particulates. The Y1 binding of neuropeptide Y was also less sensitive to chaotropic agents and guanine nucleotides than the binding of peptide YY, indicating a larger stability for association of neuropeptide Y with the receptor. Inhibition of forskolin-stimulated adenylyl cyclase showed a distinctly attenuating agonism for neuropeptide Y, with an activity similar to peptide YY below 1 nM, but considerably lower above 3 nM of the peptides. This activity was largely exerted via pertussis toxin-sensitive G-proteins of Y1-CHO cells. Our findings indicate that signaling by neuropeptide Y via its Y1 receptor could be self-restricting at higher levels of the peptide, in relation to a strong association of the agonist with the Y1 binding site.

Conformational Properties of Neuromedin NmU-8 and Its Modified Analogs

The comparative study of the spatial organization and conformational properties of NmU-8 neuropeptide and its modified analogs with available experimental data has been carried out. The effect of amino acids point mutation on conformational states of native neuropeptide has been discussed. The low-energy conformations responsible for neuropeptide contractile activity was revealed.

Flatworm nerve–muscle: structural and functional analysis

Platyhelminthes occupy a unique position in nerve–muscle evolution, being the most primitive of metazoan phyla. Essentially, their nervous system consists of an archaic brain and associated pairs of longitudinal nerve cords cross-linked as an orthogon by transverse commissures. Confocal imaging reveals that these central nervous system elements are in continuity with an array of peripheral nerve plexuses which innervate a well-differentiated grid work of somatic muscle as well as a complexity of myofibres associated with organs of attachment, feeding, and reproduction. Electrophysiological studies of flatworm muscles have exposed a diversity of voltage-activated ion channels that influence muscle contractile events. Neuronal cell types are mainly multi- and bi-polar and highly secretory in nature, producing a heterogeneity of vesicular inclusions whose contents have been identified cytochemically to include all three major types of cholinergic, aminergic, and peptidergic messenger molecules. A landmark discovery in flatworm neuro biology was the biochemical isolation and amino acid sequencing of two groups of native neuropeptides: neuro peptide F and FMRFamide-related peptides (FaRPs). Both families of neuropeptide are abundant and broadly distributed in platyhelminths, occurring in neuronal vesicles in representatives of all major flatworm taxa. Dual localization studies have revealed that peptidergic and cholinergic substances occupy neuronal sets separate from those of serotoninergic components. The physiological actions of neuronal messengers in flatworms are beginning to be established, and where examined, FaRPs and 5-HT are myoexcitatory, while cholinomimetic substances are generally inhibitory. There is immunocytochemical evidence that FaRPs and 5-HT have a regulatory role in the mechanism of egg assembly. Use of muscle strips and (or) muscle fibres from free-living and parasitic flatworms has provided baseline information to indicate that muscle responses to FaRPs are mediated by a G-protein-coupled receptor, and that the signal transduction pathway for contraction involves the second messengers cAMP and protein kinase C.

Effect of Somatostatin and Octreotide on Proliferation and Vascular Endothelial Growth Factor Secretion from Murine Endothelial Cell Line (HECa10) Culture

Angiogenesis, development of new blood vessels, is required for normal tissue repair and also for tumor cell proliferation, extracellular matrix invasion, and hematogenous metastases. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that has been shown to play a key role in neovascularization. Inhibition of angiogenesis in vitro and in vivo was documented by administration of native neuropeptide somatostatin and its analog octreotide. We have studied the effect of somatostatin-14 (SRIF) and ocreotide (sandostatin) on proliferation activity and VEGF release from cultured murine endothelial cells HECa10 in vitro. SRIF in concentrations from 10−9 to 10−5 M and ocreotide in concentrations from 10−9 to 10−5 M diminished the proliferative activity of cultured cells vs controls. SRIF and ocreotide in concentrations from 10−14 to 10−6 M did not change the release of VEGF into supernatants of 24 or 72 h endothelial cell cultures. Although we showed the antiproliferative effect of SRIF and ocreotide on mouse endothelial cells, we were unable to demonstrate the inhibitory effect of tested peptides on VEGF secretion in vitro.

Relationship between musculature and nervous system in the regenerating pharynx in Girardia tigrina (Plathelminthes)

Summary The study shows that the regenerating pharynx of Girardia tigrina forms a simple and valuable model system for studies of pattern formation in the nervous system and its relationship to the developing muscles. Immunocytochemistry was used, with antisera raised against 5-HT and the native planarian neuropeptide GYIRFamide. We studied the sequential development of the innervation in the regenerating pharynx, and using TRITC-labelled phalloidin we followed the corresponding differentiation and maturation of pharynx musculature. The peptidergic and aminergic neuronal cell types develop according to different time schedules and different modes. Throughout the process, the GYIRFamide-IR elements are in contact with the old parts of the nervous system, while the apical 5-HT-IR elements develop de novo. In the regenerating pharynx the GYIRFamide-IR nerves develop in a proximodistal direction. The first 5-HT-IR cell bodies appear in the tip of the pharynx and are symmetrically placed. They have no contact to the rest of the nervous system. From these cell bodies processes grow disto-proximally and fasciculate with fibres from the proximal part. A striking parallelism between the appearance of GYIRFamide-IR nerves and muscle fibres stained with TRITC-phalloidin was observed. The GYIRFamide-IR nerves cling to the muscle fibres. These results suggest that the contact between muscle fibres and GYIRF-IR nerves is essential for the function of pharynx. The delayed appearance of 5-HT-IR nervous elements is discussed in terms of the influence of 5-HT on sprouting of nerve fibres and synapse formation. The development of the pharynx in tail parts after fission is compared with that after pharynx amputation. The faster rate observed for pharynx regenerating after amputation in comparison with that in regenerating tail parts indicates the importance of the remains of the old nervous system in the pharynx structure.

Transglutaminase-synthesized spermine derivative of substance P recognizes rat portal vein neurokinin-3 receptors.

The effects of the transglutaminase-synthesized polyamine derivatives of Substance P (SP) have been further characterized by their ability to contract in vitro the rat portal vein strip (RPV), a pharmacological preparation particularly rich in NK-3 receptors. The effects of selective agonists of NK-1, NK-2 and NK-3 receptors [Sar9,Met(O(2))11]SP, beta-Ala8 NKA(4-10), and senktide respectively, were also evaluated by measuring RPV concentration-response curves. Peptide [GR-82334 (NK-1) and MEN-10,376 (NK-2)] and nonpeptide [WIN 51,708 (NK-1) and SR 142801 (NK-3)] NK receptor antagonists were used to confirm the participation of the different NK receptors to contractile response. Our results demonstrated that the spermine derivative of SP (Spm-SP), previously shown to be unable to recognize NK-1 and NK-2 receptors in some bioassays, contracts RPV (EC50 = 588 nM) better than the native neuropeptide (EC50 = 1120 nM). A pretreatment with thiorphan, an inhibitor of neutral endopeptidases, significantly reduced such a difference. While this inhibitor shifts the SP concentration-response curves to the left (EC50 = 720 nM) the action of Spm-SP and [Sar9,Met(O(2))11]SP were completely thiorphan-resistant. In the absence of thiorphan we found the following rank order of potency: senktide > > beta-Ala8 NKA(4-10) > [Sar9,Met(O(2))11]SP = Spm-SP > SP. Among the mentioned NK receptor antagonists, only the selective NK-3 receptor antagonist, SR 142801, shifted to the right Spm-SP and [Sar9,Met(O(2))11]SP concentration-response curve, showing pKB values of 5.84 and 5.88, respectively. Therefore, the reported results suggest that the introduction of a Spm moiety into the SP alters the parent peptide molecule by increasing its affinity for NK-3 receptors and/or by preventing its degradation by some proteolytic enzymes.